Tylactone

ABSTRACT

Tylactone (20-dihydro-20,23-dideoxytylonolide), which has the formula: ##STR1## and specified acyl ester derivatives thereof are useful intermediates in the preparation of macrolide antibiotics.

This application is a continuation of application Ser. No. 162,976,filed July 2, 1980, now abandoned.

SUMMARY OF THE INVENTION

This invention relates to a new macrolide compound and to relatedderivatives from which useful antibiotics, such as tylosin and tylosinderivatives, can be prepared. This new compound, which is20-dihydro-20,23-dideoxytylonolide, will be called tylactone forconvenience herein. Tylactone has structure 1: ##STR2## Relatedtylactone derivatives have structure 2: ##STR3## wherein R and R₁ =anacyl moiety.

The compounds of structures 1 and 2 are useful intermediates from which16-membered macrolide antibiotics can be prepared. Although nostereochemical assignments are indicated in the structures given herein,the stereochemistry of the compounds is identical to that of tylosin.

DESCRIPTION OF THE DRAWING

The infrared absorption spectrum of tylactone in chloroform is presentedin the accompanying drawing.

DETAILED DESCRIPTION

The following paragraphs describe the properties of tylactone.

Tylactone

The structure of tylactone is shown in formula 1. Tylactone is a whitesolid which crystallizes from hexane or ethyl acetate-hexane and whichmelts at about 162°-163° C. It has the following approximate percentageelemental composition: carbon, 70%; hydrogen, 9.7%; oxygen, 20.3%. Ithas an empirical formula of C₂₃ H₃₈ O₅ and a molecular weight of about394.

The infrared absorption spectrum of tylactone in chloroform is shown inthe accompanying drawing. Observable absorption maxima occur at thefollowing frequencies (cm⁻¹): 3534 (medium), 2924 (strong), 2398 (weak),2353 (weak), 1709 (very stong), 1678 (very strong), 1626 (small), 1592(very strong), 1458 (strong), 1441 (shoulder), 1404 (strong), 1379(small), 1316 (strong), 1284 (medium), 1181 (very strong), 1143(strong), 1103 (medium), 1078 (medium), 1049 (very small), 1025(medium), 984 (very strong), 958 (strong), 923 (medium), 911 (shoulder),859 (small), 868 (medium), 840 (medium), 820 (very small) and 661(small).

The ultraviolet (UV) absorption spectrum of tylactone in neutral ethanolexhibits an absorption maximum at about 282 nm (E_(1cm) ^(1%) =560).

Tylactone has the following specific rotation:

    [α].sub.D.sup.25 -55.23° (c 1, CH.sub.3 OH).

Electrometric titration of tylactone in 66% aqueous dimethylformamideindicates it has no titratable groups.

Tylactone is nearly insoluble in water, but is soluble in organicsolvents such as acetone, methanol, ethanol, dimethylformamide,chloroform, diethyl ether, petroleum ether, benzene and dimethylsulfoxide.

Tylactone can be distinguished from tylosin by silica-gel thin-layerchromatography. Sulfuric acid spray, either concentrated or dilute(50%), may be used for detection. With this detection system tylactoneappears initially as a yellow-to-brown spot. If silica-gel plates with afluorescent background are used in the chromatography, UV detection isconvenient. The approximate Rf values of tylactone are summarized inTable 1.

                  TABLE 1                                                         ______________________________________                                        Thin-Layer Chromatography of Tylactone.sup.a                                                  Rf Value                                                      Compound          A.sup.b                                                                              B                                                    ______________________________________                                         Tylactone        0.50   0.62                                                 Tylosin           0.0    0.0                                                  ______________________________________                                         .sup.a Medium: Silica gel                                                     .sup.b Solvent: A = benzene: ethyl acetate (4:1) B = benzene: ethyl           acetate (3:2)                                                            

Ester Derivatives

Tylactone can be esterified at the 3- and 5-hydroxyl groups to give acylester derivatives by treatment with acylating agents using methods knownin the art. The acyl ester derivatives of tylactone are useful asintermediates in the preparation of new macrolide antibiotics.

Typical acylating agents include anhydrides, halides (usually incombination with a base or other acid scavenger) and active esters oforganic acids. Acylation can also be achieved by using a mixture of anorganic acid and a dehydrating agent such asN,N'-dicyclohexylcarbodiimide. Acylations can also be carried outenzymatically using procedures such as those described by Okamoto et al.in U.S. Pat. No. 4,092,473. Once formed, the acyl derivatives can beseparated and purified by known techniques.

The derivatives can be prepared by esterification techniques generallyknown in the art, such as for example, treatment of the compound with astoichiometric quantity (or a slight excess) of an acylating agent, suchas an acyl anhydride, in an organic solvent (for example, pyridine) atabout 0° C. to about room temperature for from about 1 to about 24 hoursuntil esterification is substantially complete. The ester derivative canbe isolated from the reacton mixture by standard procedures such asextraction, chromatography and crystallization.

Useful esters are those of organic acids including aliphatic,cycloaliphatic, aryl, aralkyl, heterocyclic carboxylic, sulfonic andalkoxycarbonic acids of from 1 to 18 carbon atoms, and of inorganicacids, such as sulfuric and phosphoric acids.

Representative suitable esters include those derived from acids such asformic, acetic, chloroacetic, propionic, butyric, isovaleric,glucuronic, alkoxycarbonic, stearic, cyclopropanecarboxylic,cyclohexanecarboxylic, β-cyclohexylpropionic, 1-adamantanecarboxylic,benzoic, phenylacetic, phenoxyacetic, mandelic and 2-thienylaceticacids, and alkyl-, aryl-, and aralkyl-sulfonic acids, the aryl- andaralkyl- acids optionally bearing substituents such as halogen, nitro,lower alkoxy and the like on the aromatic moiety. Suitable esters alsoinclude hemiesters derived from dicarboxylic acids such as succinic,maleic, fumaric, malonic and phthalic acids.

Preparation of Tylactone

Tylactone is prepared by culturing a strain of Streptomyces fradiaewhich produces this compound under submerged aerobic conditions in asuitable culture medium until a substantial amount of the compound isproduced.

The culture medium used to grow the Streptomyces fradiae can be any oneof a number of media. For economy in production, optimal yield, and easeof product isolation, however, certain culture media are preferred.Thus, for example, preferred carbon sources in large-scale fermentationinclude carbohydrates such as dextrin, glucose, starch, and corn mealand oils such as soybean oil. Preferred nitrogen sources include cornmeal, soybean meal, fish meal, amino acids and the like. Among thenutrient inorganic salts which can be incorporated in the culture mediaare the customary soluble salts capable of yielding iron, potassium,sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate,nitrate, and like ions.

Essential trace elements necessary for the growth and development of theorganism should also be included in the culture medium. Such traceelements commonly occur as impurities in other constituents of themedium in amounts sufficient to meet the growth requirements of theorganism. It may be necessary to add small amounts (i.e. 0.2 ml/L) of anantifoam agent such as polypropylene glycol (M.W. about 2000) tolarge-scale fermentation media if forming becomes a problem.

For production of substantial quantities of tylactone submerged aerobicfermentation in tanks is preferred. Small quantities of tylactone may beobtained by shake-flask culture. Because of the time lag in productioncommonly associated with inoculation of large tanks with the spore formof the organism, it is preferable to use a vegetative inoculum. Thevegetative inoculum is prepared by inoculating a small volume of culturemedium with the spore form or mycelial fragments of the organism toobtain a fresh, actively growing culture of the organism. The vegetativeinoculum is then transferred to a larger tank. The medium used for thevegetative inoculum can be the same as that used for largerfermentations, but other media can also be used.

A preferred method of preparing tylactone is that disclosed by RichardH. Baltz and Eugene T. Seno in a co-pending patent application entitledPROCESS FOR PREPARING TYLACTONE, Ser. No. 162,977 filed July 2, 1980.That method comprises culturing a new microorganism which was obtainedby chemical mutagenesis of a Streptomyces fradiae strain which producedtylosin. The microorganism obtained by mutagenesis produces only minimalamounts of tylosin, but produces tylactone as a major component.

The new microoganism which produces tylactone is classified as a strainof Streptomyces fradiae. A culture of this microorganism has beendeposited and made part of the stock culture collection of the NorthernRegional Research Center, Agricultural Research, North Central Region,1815 North University St., Peoria, Ill., 61604, from which it isavailable to the public under the accession number NRRL 12188.

As is the case with other organisms, the characteristics of Streptomycesfradiae NRRL 12188 are subject to variation. For example, recombinants,mutants or variants of the NRRL 12188 strain may be obtained bytreatment with various known physical and chemical mutagens, such asultraviolet light, X-rays, gamma rays, andN-methyl-N'-nitro-N-nitrosoguanidine. All natural and induced variants,mutants and recombinants of Streptomyces fradiae NRRL 12188 which retainthe characteristic of tylactone production may be used to prepare thecompounds of this invention.

S. fradiae NRRL 12188 can be grown at temperatures between about 10° andabout 40° C. Optimum production of tylactone appears to occur attemperatures of about 28° C.

As is customary in aerobic submerged culture processes, sterile air isbubbled through the culture medium. For efficient antibiotic productionthe percent of air saturation for tank production should be about 30% orabove (at 28° C. and one atmosphere of pressure).

Production of tylactone can be followed during the fermentation bytesting samples of the broth, using high-performance liquidchromatography with a UV detection system [see, for example, J. H.Kennedy in J. Chromatographic Science, 16, 492-495 (1978)].

Following its production under submerged aerobic fermentationconditions, tylactone can be recovered from the fermentation medium bymethods used in the fermentation art. Because of the limited solubilityof tylactone in water, it may not be altogether soluble in the medium inwhich it is produced. Recovery of tylactone, therefore, can beaccomplished by (1) extraction of the fermentation broth of (2)filtration of the fermentation broth and extraction of both the filteredbroth and the mycelial cake. A variety of techniques may be used in theextraction processes. A preferred technique for purification of thefiltered broth involves extracting the broth (generally without pHadjustment) with a suitable solvent such as amyl acetate or petroleumether, concentrating the organic phase under vacuum to give crystals oran oil. If an oil is obtained, it may be purified by adsorptionchromatography.

The compounds of structures 1 and 2 are useful intermediates from which16-membered macrolide antibiotics can be prepared. For example,tylactone (1) can be bioconverted to tylosin by adding it to a growingculture of a bioconverting microorganism. The bioconvertingmicroorganism can be a Streptomyces strain which either produces tylosinitself or is capable of producing tylosin except that it is blocked intylactone formation.

A strain which is capable of producing tylosin except that it is blockedin tylactone formation can be obtained by treating a tylosin-producingstrain with a mutagen and screening survivors for those which are unableto produce tylosin. Those survivors which are unable to produce tylosinare further screened to determine which strains are also unable toproduce tylactone. These strains are identified by adding tylactone tosmall shake-flask cultures of the selected survivors to determine ifthey produce tylosin.

Streptomyces fradiae strains NRRL 2702 and NRRL 2703 are examples ofStreptomyces strains which are capable of producing tylosin. A typicalmutagen which may be used to obtain the selected strains isN-methyl-N'-nitro-nitrosoguanidine.

The compound of structure 1 is especially useful in the preparation oflabeled compounds for metabolic studies. By labeling either thetylactone portion or the added sugar moieties, the metabolic pathway oftylosin can be ascertained.

In order to illustrate more fully the operation of this invention, thefollowing examples are provided:

EXAMPLE 1 A. Shake-flask Fermentation of Tylactone

A lyophilized pellet of Streptomyces fradiae NRRL 12188 is dispersed in1-2 ml of sterilized water. A portion of this solution (0.5 ml) is usedto inoculate a vegetative medium (150 ml) having the followingcomposition:

    ______________________________________                                        Ingredient       Amount (%)                                                   ______________________________________                                        Corn steep liquor                                                                              1.0                                                          Yeast extract    0.5                                                          Soybean grits    0.5                                                          CaCO.sub.3       0.3                                                          Soybean oil (crude)                                                                            0.45                                                         Deionized water  97.25                                                        ______________________________________                                    

Alternatively, a vegetative culture of S. fradiae NRRL 12188 preserved,in 1-ml volumes, in liquid nitrogen is rapidly thawed and used toinoculate the vegetative medium. The inoculated vegetative medium isincubated in a 500-ml Erlenmeyer flask at 29° C. for about 48 hours on aclosed-box shaker at about 300 rpm.

This incubated vegetative medium (0.5 ml) is used to inoculate 7 ml of aproduction medium having the following composition:

    ______________________________________                                        Ingredient       Amount (%)                                                   ______________________________________                                        Beet molasses    2.0                                                          Corn meal        1.5                                                          Fish meal        0.9                                                          Corn gluten      0.9                                                          NaCl             0.1                                                          (NH.sub.4).sub.2 HPO.sub.4                                                                     0.04                                                         CaCO.sub.3       0.2                                                          Soybean oil (crude)                                                                            3.0                                                          Deionized water  91.36                                                        ______________________________________                                    

The inoculated fermentation medium is incubated in a 50-ml bottle at 29°C. for about 6 days on a closed-box shaker at 300 rpm.

B. Tank Fermentation of Tylactone

In order to provide a larger volume of inoculum, 60 ml of incubatedvegetative medium, prepared in a manner similar to that described insection A, is used to inoculate 38 L of a second-stage vegetative growthmedium having the following compositions:

    ______________________________________                                        Ingredient       Amount (%)                                                   ______________________________________                                        Corn steep liquor                                                                              1.0                                                          Soybean meal     0.5                                                          Yeast extract    0.5                                                          CaCO.sub.3       0.3                                                          Soybean oil (crude)                                                                            0.5                                                          Lecithin (crude) 0.015                                                        Water            97.185                                                       ______________________________________                                    

Adjust pH to 8.5 with 50% NaOH solution.

This second-stage vegetative medium is incubated in a 68-liter tank forabout 47 hours at 29° C.

Incubated second-stage medium (4 L) thus prepared is used to inoculate40 liters of sterile production medium having the following composition:

    ______________________________________                                        Ingredient       Amount (%)                                                   ______________________________________                                        Fish meal        0.92                                                         Corn meal        1.57                                                         Corn gluten      0.92                                                         CaCO.sub.3       0.21                                                         NaCl             0.10                                                         (NH.sub.4).sub.2 HPO.sub.4                                                                     0.04                                                         Beet molasses    2.10                                                         Soybean oil (crude)                                                                            3.15                                                         Lecithin         0.09                                                         Water            90.90                                                        ______________________________________                                    

Adjust pH to 7.2 with 50% NaOH solution.

The inoculated production medium is allowed to ferment in a 68-litertank for about 5 days at a temperature of 28° C. The fermentation mediumis aerated with sterile air to keep the dissolved oxygen level betweenabout 30% and 50% and is stirred with conventional agitators at about300 rpm.

EXAMPLE 2 Isolation of Tylactone

Fermentation broth (1600 L), obtained as described in Example 1, isfiltered using a filter aid (3% Hyflo Supercel, a diatomaceous earth,Johns Manville Corp.). The pH of the filtrate is adjusted to about 9 bythe addition of 2% sodium hydroxide. The filtrate is extracted with amylacetate (400 L). The amyl acetate extract (which has a high opticaldensity reading at 282 nm but no antimicrobial activity) is concentratedunder vacuum to give an oil. The oil is dissolved in benzene (5 L). Thebenzene solution is chromatographed over a 5.25-×36-in. silica-gel(Grace, grade 62, Davison Chemical Co.) column, packed with benzene.Elution is monitored by silica-gel thin-layer chromatography, using abenzene:ethyl acetate (3:2) solvent system and conc. sulfuric acid sprayfor detection. The column is first eluted with benzene to remove lipidsubstances, then with benzene:ethyl acetate (9:1) to separate andisolate tylactone. Fractions containing tylactone are combined andevaporated under vacuum. Tylactone is crystallized from benzene-hexaneor hot hexane to give about 2 g, m.p. 162°-163° C.

EXAMPLE 3 3,5-Di-O-acetyltylactone

Tylactone (200 mg), prepared as described in Example 2, is dissolved inpyridine (4 ml). Acetic anhydride (4 ml) is added. The resulting mixtureis allowed to stand at room temperature for 16 hours and then isconcentrated to dryness under vacuum. Methanol (5 ml) is added to theresidue; the solution is heated at 60° for 1/2 hour and then isconcentrated under vacuum to give 3,5-di-O-acetyltylactone. Thiscompound has an R_(f) value of about 0.59 on silica-gel thin-layerchromatography in a benzene:ethyl acetate (4:1) solvent system. TheR_(f) of tylactone in this system is about 0.3.

EXAMPLES 4-7

3,5-Di-O-Propionyltylactone, prepared according to the procedure ofExample 3, but using propionic anhydride.

3,5-Di-O-isovaleryltylactone, prepared according to the procedure ofExample 3, but using isovaleric anhydride.

3,5-Di-O-benzoyltylactone, prepared according to the procedure ofExample 3, but using benzoic anhydride.

3,5-Di-O-(n-butyryl)tylactone, prepared according to the procedure ofExample 3, but using n-butyric anhydride.

EXAMPLE 8 Preparation of Tylosin from Tylactone

A Streptomyces fradiae strain which formerly produced tylosin but whichis blocked in macrolide ring closure is fermented according to theprocedure described in Example 1, Section A, except that a temperatureof 28° C. is used. Tylactone is added to the fermentation 48 hours afterinoculation. The fermentation is then continued until a substantialamount of tylosin is produced, i.e. about three additional days. Thepresence of tylosin is determined by testing samples of the brothagainst organisms known to be sensitive to tylosin. One useful assayorganism is Staphylococcus aureus ATCC 9144. Bioassay is convenientlyperformed by an automated turbidometric method, by thin-layerchromatography or by high-performance liquid chromatography with UVdetection.

EXAMPLE 9

Tylactone is prepared by the method of Example 1 except that a labeledacetate, propionate, or butyrate is incorporated into the fermentationmedium. Labeled tylactone thus produced is used to prepare tylosinaccording to the procedure of Example 8. Tylosin labeled on themacrolide ring is thereby provided.

EXAMPLE 10

Tylactone, prepared by the method of Example 1, is used to preparetylosin according to the method of Example 8 except that a labeled sugarmoiety such as glucose is added to the second fermentation to providetylosin which is labeled on the sugar moiety.

We claim:
 1. Tylactone which has the structure: ##STR4## and the diacylester derivative of tylactone wherein each is an ester of amonocarboxylic acid or a hemi-ester of a dicarboxylic acid, each of 1 to18 carbon atoms.
 2. The compound of claim 1 which is tylactone.
 3. Thecompound of claim 1 which is the diacyl ester derivative of tylactone.4. The compound of claim 3 which is 3,5-di-O-acetyltylactone.
 5. Thecompound of claim 3 which is 3,5-di-O-propionyltylactone.
 6. Thecompound of claim 3 which is 3,5-di-O-benzoyltylactone.
 7. The compoundof claim 3 which is 3,5-di-O-isovaleryltylactone.
 8. The compound ofclaim 3 which is 3,5-di-O-(n-butyryl)tylactone.